Validation of Reference Genes for Robust qRT-PCR Gene Expression Analysis in the Rice Blast Fungus Magnaporthe oryzae.
Validation of Reference Genes for Robust qRT-PCR Gene Expression Analysis in the Rice Blast Fungus Magnaporthe oryzae.
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水稻疫霉菌 Magnaporthe oryzae 中用于稳健 qRT-PCR 基因表达分析的参考基因验证
DOI：
10.1371/journal.pone.0160637
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Gurr SJ
中科院分区：
文献类型：
--
作者：
Che Omar S;Bentley MA;Morieri G;Preston GM;Gurr SJ
稻瘟病菌每年造成重大的收成损失。它也可以作为一个遗传学上易于处理的模型来研究真菌入侵。虽然致病性决定因素已被揭露，并在全球基因表达的变化描述，我们所知甚少的稻瘟病菌细胞壁重塑。我们的兴趣，在感染，营养生长和外源性壁应力下表达的壁重塑基因，需要强大的选择参考基因的定量真实的时间-PCR（qRT-PCR）数据标准化。我们描述了9个候选参考基因的表达稳定性，通过qRT-PCR与来自无性生殖发育过程中，从性阶段perithelium和营养菌丝生长在各种外源性压力下的cDNA。我们的qRT-PCR实验（MIQE）合规分析的最小信息公布揭示了一组用于跟踪细胞壁重塑基因MGG_Crh 2（MGG_00592）表达变化的稳健参考基因。我们根据表达稳定性（M）对9个候选参考基因进行了排名，并报告了在M中常用的3个组织组（感染性、营养性和全局）中进行检测时，可靠基因表达正常化所需的最佳基因组合。表达研究。我们发现MGG_Actin（MGG_03982）和40 S 27 a核糖体亚基MGG_40s（MGG_02872）被证明是感染组的稳健参考基因，而MGG_40s和MGG_Ef1（Elongation Factor 1-α）被证明是营养组和全局组的稳健参考基因。利用上述已验证的参考基因，M.发现在营养生长期间，与休眠孢子相比，MGG_Crh 2表达显著（p<0.05）升高三倍，并且与在最佳条件下生长相比，在细胞壁胁迫（刚果红）下，MGG_Crh 2表达高两倍。我们建议组合使用属于细胞骨架和核糖体合成功能组的两个参考基因，MGG_Actin、MGG_40s、MGG_S8（核糖体亚基40 S S8）或MGG_Ef1，它们在异质组织中表现出低M值。相比之下，代谢途径基因MGG_Fad（含FAD结合结构域的蛋白质）和MGG_Gapdh（甘油醛-3-磷酸脱氢酶）表现较差，这是由于它们在样品间缺乏表达稳定性。
The rice blast fungus causes significant annual harvest losses. It also serves as a genetically-tractable model to study fungal ingress. Whilst pathogenicity determinants have been unmasked and changes in global gene expression described, we know little about Magnaporthe oryzae cell wall remodelling. Our interests, in wall remodelling genes expressed during infection, vegetative growth and under exogenous wall stress, demand robust choice of reference genes for quantitative Real Time-PCR (qRT-PCR) data normalisation. We describe the expression stability of nine candidate reference genes profiled by qRT-PCR with cDNAs derived during asexual germling development, from sexual stage perithecia and from vegetative mycelium grown under various exogenous stressors. Our Minimum Information for Publication of qRT-PCR Experiments (MIQE) compliant analysis reveals a set of robust reference genes used to track changes in the expression of the cell wall remodelling gene MGG_Crh2 (MGG_00592). We ranked nine candidate reference genes by their expression stability (M) and report the best gene combination needed for reliable gene expression normalisation, when assayed in three tissue groups (Infective, Vegetative, and Global) frequently used in M. oryzae expression studies. We found that MGG_Actin (MGG_03982) and the 40S 27a ribosomal subunit MGG_40s (MGG_02872) proved to be robust reference genes for the Infection group and MGG_40s and MGG_Ef1 (Elongation Factor1-α) for both Vegetative and Global groups. Using the above validated reference genes, M. oryzae MGG_Crh2 expression was found to be significantly (p<0.05) elevated three-fold during vegetative growth as compared with dormant spores and two fold higher under cell wall stress (Congo Red) compared to growth under optimal conditions. We recommend the combinatorial use of two reference genes, belonging to the cytoskeleton and ribosomal synthesis functional groups, MGG_Actin, MGG_40s, MGG_S8 (Ribosomal subunit 40S S8) or MGG_Ef1, which demonstrated low M values across heterogeneous tissues. By contrast, metabolic pathway genes MGG_Fad (FAD binding domain-containing protein) and MGG_Gapdh (Glyceraldehyde-3-phosphate dehydrogenase) performed poorly, due to their lack of expression stability across samples.